霍乱肠毒素ctxA基因真核表达重组质粒的构建及表达

目的 构建霍乱肠毒素A亚单位基因（ctxA）真核表达重组质粒，并在NIH3T3细胞中进行表达。方法 用限制性核酸内切酶从重组质粒pET32a—ctxA上切下ctxA基因，导入真核表达载体peDNA3．1（＋）．重组子pcDNA3．1-ctxA经限制性酶切分析、PCR鉴定和序列测定正确后，用脂质体法将重组质粒pcDNA3．1-ctxA转染NIH3T3细胞，采用免疫荧光法和Westernblot对peDNA3．1-ctxA的瞬时表达产物和稳定表达产物进行鉴定。结果 重组质粒peD—NA3．1-ctxA成功转入NIH3T3细胞。利用免疫荧光技术在NIH3T3细胞膜和细胞浆中检测到了瞬时表达，用Westernblot检测到转染的阳性细胞克隆稳定表达出约29ku的蛋白。结论 成功构建霍乱肠毒素A亚单位基因真核表达重组质粒peDNA3．1-ctxA，并在NIH3T3细胞中表达出29ku的CTA蛋白。

Construction and expression of the eukaryotic recombinant plasmid of ctxA gene of Vibrio cholerae

Objective To construct recombinant plasmid pcDNA3.1-ctxA and detect the expression of pcDNA3.1-ctxA in NIH3T3 cell.Methods The ctxA gene was obtained from pET32a-ctxA by restriction endonuclease digestion and was subcloned into eukaryotic expressed vector pcDNA3.1(+).The recombinant plasmid,which was named pcDNA3.1-ctxA,was identified by restriction analysis,PCR and DNA sequencing analysis.NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-ctxA with Lipofection strategy.Transient and stable products of ctxA gene was detected by immunofluorescence and Western blot.Results The recombinant plasmid pcDNA3.1-ctxA was detected its expression in NIH3T3 cell membrane and cytoplasm with immunofluorescence.There was the 29 ku protein in stable transfection NIH3T3 cells by Western blot analysis.Conclusion The recombinant plasmid pcDNA3.1-ctxA was successfully constructed and 29 ku CTA protein is expressed in NIH3T3 cells.